The overall goal of these studies is to determine the mechanism(s) underlying stress-induced immune suppression. Stress has significant effects on the immune system of humans, rodents, and monkeys. Previous studies support the hypothesis that stressors modulate immune regulation through stress hormones such as endogenous opioids, other than exclusively glucocorticoids. However, the mechanisms by which stress affect the immune and neuroendocrine systems remain to be established. Our recent studies revealed that restraint stress of mice induces CD95-mediated apoptosis in splenocytes in an endogenous opioid-dependent manner. Our preliminary data showed that inhibition of phosphatidylinositol 3-kinase (PI3K) and nuclear factor kappaB (NF-kappaB) signaling exerts an additive effect on stress-induced splenocyte reduction. In addition, we have also shown that inhibition of p53 partially blocks stress-induced lymphocyte reduction in the spleen; and that opioid receptor antagonists inhibit the stress-induced increase in p53 expression. Our overall hypothesis is that increased production of endogenous opioids is critical to immune suppression during stress. Moreover, we postulate that the mechanism involves the anti-apoptotic PI3K/NF-kappaB pathway and the pro-apoptotic p53 pathway. The first specific aim is to study the contributions of PI3K and NF-kappaB in opioids-mediated immune suppression. Mice will be treated with or without PI3K/NF-kappaB inhibitors followed by treatment with or without opioid receptor antagonists, and then subjected to physical restraint stress for different time periods. These studies will emphasize an analysis of lymphocyte number, response to mitogenic stimulation, cytokine production, and the level of endogenous opioids. We intend to give particular attention to examine the effects of endogenous opioids and PI3K/NF-kappaB on immune responses in vivo. We also propose studies, as the second specific aim, to determine the role of p53 in opioids-mediated lymphocyte reduction. We will use a p53 inhibitor and p53 knockout mice to determine the role of p53 in physical restraint stress. Our emphasis will be on lymphocyte number, the number of apoptotic cells, p53 expression, and the expression of CD95 and CD95 ligand. These studies should lead to our understanding of the effects of a stress environment on the immune and psychoneuroendocrine systems.